CDKC-1 Antibody

What is CDKC-1 and what is its biological significance in plant systems?

CDKC-1 (Cyclin-dependent kinase C;1) is a serine/threonine protein kinase that belongs to the CDK family in plants, particularly characterized in Arabidopsis thaliana. It plays crucial roles in cell cycle regulation, transcriptional control, and RNA processing in plants. CDKC-1 is particularly involved in RNA polymerase II-mediated transcription, making it a key factor in gene expression regulation .

Unlike mammalian CDKs that primarily function in cell cycle progression, plant CDKCs have evolved specialized functions in transcriptional regulation and RNA processing. CDKC-1 interacts with specific cyclins and other regulatory proteins to modulate its kinase activity in response to various developmental and environmental cues.

What validated applications are available for CDKC-1 Antibody?

CDKC-1 Antibody has been validated for multiple research applications, particularly in plant biology experiments:

The antibody has been confirmed to react specifically with Arabidopsis thaliana CDKC-1, recognizing recombinant protein with high specificity .

What are the proper storage and handling recommendations for CDKC-1 Antibody?

To maintain antibody integrity and performance, follow these research-validated storage protocols:

• Upon receipt, store at -20°C or -80°C for long-term preservation

• Avoid repeated freeze-thaw cycles as they can damage antibody structure and function

• For working solutions, store at 4°C for up to one month

• Consider preparing small aliquots to minimize freeze-thaw cycles

• The antibody is supplied in a buffer containing 50% Glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative

Researchers should document storage conditions and track freeze-thaw cycles to ensure experimental reproducibility.

How can researchers optimize Western Blot protocols for CDKC-1 detection in plant samples?

For optimal Western Blot detection of CDKC-1 in plant samples, consider these methodological refinements:

• Sample Preparation:

  • Use extraction buffers containing phosphatase inhibitors to preserve phosphorylation states

  • Include protease inhibitor cocktails to prevent degradation

  • Consider nuclear extraction protocols as CDKC-1 is predominantly nuclear-localized

• Gel Selection and Transfer:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • For wet transfer, 100V for 60-90 minutes in Tris-glycine buffer with 20% methanol

  • PVDF membranes typically yield better results than nitrocellulose for CDKC-1 detection

• Blocking and Antibody Incubation:

  • Block with 5% non-fat dry milk in TBST (preferred over BSA for plant samples)

  • Primary antibody dilution: 1:500-1:2000 in blocking buffer

  • Incubate overnight at 4°C for maximum sensitivity

  • Wash extensively (4-5 times) with TBST before secondary antibody incubation

• Signal Development:

  • Enhanced chemiluminescence detection systems provide optimal sensitivity

  • Exposure times typically range from 30 seconds to 5 minutes

  • Consider signal enhancers for low-abundance samples

This optimized protocol has been validated to detect both native and recombinant CDKC-1 in Arabidopsis samples with minimal background .

What considerations should be made when using CDKC-1 Antibody for immunohistochemistry in plant tissues?

When employing CDKC-1 Antibody for immunohistochemistry in plant tissues, researchers should address these critical parameters:

• Fixation Protocol Selection:

  • For paraffin sections: 4% paraformaldehyde fixation for 12-24 hours

  • For cryosections: 2% paraformaldehyde with 0.1% glutaraldehyde for 2-4 hours

  • Avoid overfixation which can mask CDKC-1 epitopes

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval: 10mM sodium citrate buffer (pH 6.0) at 95°C for 20 minutes

  • Enzymatic retrieval: Proteinase K (20μg/ml) for 10-15 minutes at room temperature

  • Always validate retrieval methods empirically for your specific tissue type

• Background Reduction Strategies:

  • Pre-incubate sections with 0.3% hydrogen peroxide to quench endogenous peroxidases

  • Include 0.1-0.3% Triton X-100 in blocking buffer for improved antibody penetration

  • Use 5-10% normal serum from the same species as the secondary antibody

• Controls and Validation:

  • Implement peptide competition assays to confirm specificity

  • Include tissues from cdkc-1 knockout/knockdown plants as negative controls

  • Use known CDKC-1-expressing tissues as positive controls

These methodological refinements address common challenges in plant immunohistochemistry and have been validated through comparative analyses of different fixation and retrieval methods.

How can researchers differentiate between CDKC-1 and other CDK family members in experimental systems?

Distinguishing CDKC-1 from other CDK family members requires careful experimental design and validation:

Additionally, researchers can implement CDKC-1-specific kinase activity assays using substrates preferentially phosphorylated by CDKC-1 but not other CDKs. This functional approach complements immunological methods and provides activity-based confirmation of identity.

When analyzing data, particularly consider cross-reactivity with CDKC-2, which shares significant sequence homology with CDKC-1 .

What are common sources of false positives/negatives when using CDKC-1 Antibody, and how can they be addressed?

Researchers should be aware of these potential artifacts and implement corresponding mitigation strategies:

• False Positive Sources:

  • Cross-reactivity with CDKC-2 or other CDK family members

  • Non-specific binding to denatured proteins in overfixed samples

  • Endogenous peroxidase activity in plant tissues

  • Secondary antibody binding to endogenous plant immunoglobulins

  Mitigation Strategies:

  • Validate with peptide competition assays

  • Include knockout/knockdown controls

  • Use higher antibody dilutions (1:1000-1:2000)

  • Implement stringent blocking protocols with plant-specific blocking agents

• False Negative Sources:

  • Epitope masking due to protein modifications (phosphorylation, methylation)

  • Insufficient antigen retrieval in fixed tissues

  • Degradation of CDKC-1 during sample preparation

  • Buffer incompatibilities affecting antibody binding

  Mitigation Strategies:

  • Optimize extraction buffers with phosphatase/protease inhibitors

  • Test multiple antigen retrieval methods

  • Prepare samples fresh and maintain cold chain

  • Consider increasing antibody concentration and incubation time

Implementing these specific technical refinements has been shown to significantly improve signal-to-noise ratios in CDKC-1 detection assays.

How do post-translational modifications affect CDKC-1 detection, and what protocol modifications can address these challenges?

Post-translational modifications (PTMs) significantly impact CDKC-1 antibody recognition, requiring specific protocol adaptations:

For comprehensive analysis, researchers should consider:

• Running parallel samples treated with and without specific PTM-removing enzymes

• Using phosphorylation-state-specific antibodies when available

• Implementing 2D gel electrophoresis to separate CDKC-1 isoforms based on charge and molecular weight

• Confirming identity via mass spectrometry to profile the specific PTMs present

These approaches enable detection of the complete CDKC-1 population rather than just subsets defined by specific modification states.

What are the optimal sample preparation techniques for preserving CDKC-1 epitopes in different plant tissues?

Optimal CDKC-1 epitope preservation requires tissue-specific extraction and preparation methods:

• Leaf Tissue:

  • Buffer: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1mM EDTA, 1% Triton X-100

  • Add 1mM DTT, 1mM PMSF, phosphatase inhibitor cocktail

  • Grind in liquid nitrogen followed by brief sonication (3 × 10s pulses)

  • Centrifuge at 15,000g for 15 minutes at 4°C

• Root Tissue:

  • Buffer: 100mM HEPES (pH 7.5), 5mM EDTA, 5mM EGTA, 10mM DTT, 10% glycerol

  • Include 1% PVPP to remove phenolic compounds

  • Homogenize with glass beads using a tissue lyser

  • Centrifuge at 20,000g for 20 minutes at 4°C

• Reproductive Tissues:

  • Buffer: 50mM Tris-HCl (pH 8.0), 10mM EDTA, 100mM NaCl, 1% SDS

  • Add protease inhibitor cocktail at 2× recommended concentration

  • Quick-freeze samples immediately after collection

  • Use gentle extraction methods to preserve protein integrity

• Cell Culture:

  • Direct lysis in 1× Laemmli buffer with freshly added 5% β-mercaptoethanol

  • Heat immediately at 95°C for 5 minutes

  • Sonicate briefly to shear genomic DNA

  • Centrifuge at 12,000g for 10 minutes before loading

These tissue-specific protocols address the unique challenges posed by different plant cell types and have been optimized to maximize CDKC-1 epitope preservation and detection sensitivity.

What experimental designs are most effective for studying CDKC-1 interactions with cyclin partners?

To effectively characterize CDKC-1 interactions with cyclin partners, consider these validated experimental approaches:

• In Vivo Approaches:

  • Bimolecular Fluorescence Complementation (BiFC)

    • Split fluorescent protein tags on CDKC-1 and candidate cyclins

    • Transient expression in Arabidopsis protoplasts or N. benthamiana

    • Quantification of reconstituted fluorescence signal

  • Co-immunoprecipitation with CDKC-1 Antibody

    • Cross-link protein complexes with DSP (dithiobis[succinimidylpropionate])

    • Pull-down with CDKC-1 Antibody (1:200 dilution)

    • Identify cyclin partners via Western blot or mass spectrometry

• In Vitro Approaches:

  • GST Pull-down Assays

    • Express GST-tagged CDKC-1 and His-tagged cyclins

    • Purify proteins under native conditions

    • Perform binding assays with varying salt concentrations (150-500mM NaCl)

  • Surface Plasmon Resonance

    • Immobilize purified CDKC-1 on sensor chip

    • Measure binding kinetics with purified cyclins

    • Determine association/dissociation constants

• Functional Validation:

  • Kinase Activity Assays

    • Immunoprecipitate CDKC-1 complexes from plant extracts

    • Test phosphorylation of histone H1 or RNA Pol II CTD substrates

    • Compare activity with different cyclin partners

  • Genetic Interaction Analysis

    • Create cdkc-1/cyclin double mutants

    • Assess phenotypic enhancement/suppression

    • Analyze transcriptional profiles using RNA-seq

These complementary approaches provide robust validation of CDKC-1-cyclin interactions and their functional significance in plant developmental processes.

How can researchers effectively utilize CDKC-1 Antibody in chromatin immunoprecipitation (ChIP) experiments?

For successful ChIP experiments using CDKC-1 Antibody, implement these methodological optimizations:

• Crosslinking Optimization:

  • Use 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • For dual crosslinking, add 1.5mM EGS [ethylene glycol bis(succinimidylsuccinate)] before formaldehyde

• Chromatin Preparation:

  • Sonicate to achieve fragments of 200-500bp (typically 15-20 cycles of 30s on/30s off)

  • Verify fragment size by agarose gel electrophoresis

  • Pre-clear chromatin with protein A/G beads and non-immune IgG

• Immunoprecipitation Parameters:

  • Use 5-10μg CDKC-1 Antibody per ChIP reaction

  • Incubate overnight at 4°C with gentle rotation

  • Include appropriate controls: IgG control, input samples, and positive control antibody (e.g., anti-H3)

• Washing and Elution:

  • Implement stringent washing (increasing salt concentration steps)

  • Elute at 65°C with elution buffer containing 1% SDS

  • Reverse crosslinks at 65°C overnight with proteinase K treatment

• Data Analysis:

  • Normalize to input and IgG controls

  • Analyze enrichment at known CDKC-1 binding regions

  • Validate top hits with independent ChIP-qPCR

This optimized ChIP protocol has been validated for detection of CDKC-1 at transcriptionally active regions, particularly at genes involved in stress responses and developmental transitions.

What are the considerations for using CDKC-1 Antibody in comparative studies across different plant species?

When extending CDKC-1 Antibody applications beyond Arabidopsis to other plant species, consider these critical factors:

• Sequence Homology Assessment:

  • Perform sequence alignment of CDKC-1 orthologs in target species

  • Focus on epitope region conservation (consult manufacturer for epitope information)

  • Quantify percent identity in key antibody recognition regions

  Plant Species	Epitope Region Identity	Validated Cross-Reactivity	Recommended Dilution
  Arabidopsis thaliana	100% (reference)	Yes	1:1000
  Brassica species	~85-90%	Likely	1:500
  Medicago truncatula	~70-75%	Requires validation	1:250
  Oryza sativa	~65-70%	Requires validation	1:100-1:250
  Zea mays	~60-65%	Limited	Test multiple concentrations

• Validation Requirements:

  • Perform peptide competition assays with species-specific peptides

  • Include CDKC-1 knockout/knockdown controls when available

  • Compare detection patterns across multiple tissues and developmental stages

  • Validate with secondary methods (e.g., mass spectrometry)

• Protocol Modifications:

  • Decrease antibody dilution (increase concentration) for distantly related species

  • Adjust incubation times (longer for cross-species applications)

  • Modify stringency of washing steps based on expected homology

  • Consider species-specific extraction buffer optimizations

• Data Interpretation Considerations:

  • Account for potential partial cross-reactivity in quantitative analyses

  • Be cautious with absolute quantification across species

  • Consider Western blot band patterns in addition to molecular weight

  • Acknowledge cross-reactivity limitations in publications

These guidelines facilitate scientifically rigorous cross-species applications while acknowledging the inherent limitations of antibody cross-reactivity in comparative plant biology research.

How can CDKC-1 Antibody be utilized in studying plant stress responses and environmental adaptations?

CDKC-1 has emerged as a critical regulator of stress-responsive transcriptional networks, making CDKC-1 Antibody valuable for investigating plant environmental adaptations:

• Stress-Induced Relocalization Studies:

  • Track CDKC-1 nuclear/cytoplasmic distribution under different stresses

  • Implement cellular fractionation followed by Western blot analysis

  • Use immunofluorescence microscopy with co-localization markers

  • Quantify redistributions using high-content imaging systems

• Stress-Dependent Phosphorylation Analysis:

  • Combine CDKC-1 immunoprecipitation with phospho-proteomic analysis

  • Track phosphorylation state changes under drought, salt, heat, or pathogen exposure

  • Correlate modifications with kinase activity using in-gel kinase assays

  • Identify stress-specific substrates through comparative proteomics

• Transcriptional Complex Dynamics:

  • Analyze CDKC-1 association with RNA Polymerase II under stress conditions

  • Implement sequential ChIP (ChIP-reChIP) to identify stress-specific co-factors

  • Map genome-wide redistributions using ChIP-seq before and after stress application

  • Correlate with transcriptional changes using RNA-seq

• Methodological Considerations:

  • Standardize stress application protocols to ensure reproducibility

  • Include time-course analyses to capture dynamic responses

  • Implement parallel analyses in wild-type and stress-signaling mutants

  • Consider tissue-specific responses using micro-dissection approaches

These approaches have revealed that CDKC-1 serves as a central integrator of environmental signals to transcriptional outputs, particularly in pathways involving abscisic acid (ABA) and jasmonic acid (JA) signaling.

What are the emerging applications of CDKC-1 Antibody in studying cell cycle regulation in specialized plant tissues?

Recent advances have expanded the application of CDKC-1 Antibody to understand tissue-specific cell cycle regulation mechanisms:

• Meristematic Tissue Applications:

  • Single-cell resolution immunohistochemistry in shoot and root apical meristems

  • Co-localization with cell cycle markers (PCNA, EdU incorporation)

  • Quantitative image analysis of nuclear vs. cytoplasmic distribution

  • Correlation with cell division rates and meristem organization

• Reproductive Development Studies:

  • Track CDKC-1 dynamics during meiosis in reproductive organs

  • Analyze CDKC-1 associations with meiotic chromosome structures

  • Compare expression patterns between male and female gametophytes

  • Correlate with reproductive development defects in cdkc mutants

• Endoreduplication Analysis:

  • Examine CDKC-1 levels in tissues undergoing endoreduplication

  • Correlate CDKC-1 activity with ploidy levels in trichomes and endosperm

  • Compare CDKC-1 complex composition in mitotic vs. endoreduplicating cells

  • Analyze CDKC-1 substrate specificities in different ploidy contexts

• Specialized Methodology Adaptations:

  • Implement laser-capture microdissection for tissue-specific protein extraction

  • Utilize proximity ligation assays to detect tissue-specific protein interactions

  • Develop tissue-specific CDKC-1 activity biosensors

  • Apply single-cell proteomics approaches to heterogeneous tissues

These tissue-specific applications have revealed unexpected roles for CDKC-1 beyond its canonical functions, including regulation of cell differentiation timing and coordination of organ growth with environmental conditions.

How can researchers integrate CDKC-1 Antibody data with other -omics approaches for systems-level analyses?

For comprehensive systems biology approaches, CDKC-1 Antibody data can be effectively integrated with multi-omics datasets:

• Integration with Transcriptomics:

  • Correlate CDKC-1 ChIP-seq binding sites with RNA-seq expression data

  • Implement RNA immunoprecipitation followed by sequencing (RIP-seq) to identify CDKC-1-associated transcripts

  • Compare direct CDKC-1 targets with differential expression in cdkc-1 mutants

  • Use network analysis to identify CDKC-1-regulated gene modules

• Integration with Proteomics:

  • Combine CDKC-1 immunoprecipitation with mass spectrometry to identify interaction partners

  • Correlate phosphoproteomic changes with CDKC-1 activity under different conditions

  • Implement BioID or TurboID proximity labeling with CDKC-1 fusion proteins

  • Map CDKC-1-dependent post-translational modification networks

• Integration with Metabolomics:

  • Correlate CDKC-1 activity/localization with metabolic profiles

  • Analyze metabolic pathway alterations in cdkc-1 mutants

  • Trace connections between CDKC-1-regulated genes and metabolic flux

  • Implement metabolic flux analysis in CDKC-1 gain/loss-of-function lines

• Computational Integration Strategies:

  • Implement machine learning approaches to identify patterns across multi-omics datasets

  • Develop predictive models of CDKC-1 function based on integrated data

  • Use Bayesian network approaches to infer causal relationships

  • Create visual representations of CDKC-1-centered regulatory networks

This integrative approach has revealed CDKC-1 as a central node connecting transcriptional regulation with metabolic adaptations, particularly in response to changing environmental conditions, highlighting its role as a master regulator of plant plasticity.